Gfp monoclonal antibody

HepG2, 293T and PC3 cells were obtained from the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, Shanghai). Cells were cultured in high glucose DMEM (C11995500BT, Life Technologies) containing 10% fetal bovine serum (FBS) (10099–141, Life Technologies) with 100 units/ml penicillin and 10 μg/ml streptomycin (15140–122, Life Technologies) at 37°C in a 5% CO₂ atmosphere.
The polyclonal antibody against STAT3 C-20 (sc-482) and monoclonal antibodies for Myc (sc-40) and pY20 (sc-508) were from Santa Cruz Biotechnology, Inc. The GFP monoclonal antibody (11814460001) was from Roche and the polyclonal antibody for acetyl-lysine (#9441s) was from Cell Signaling Technology. The secondary antibodies, including goat anti-rabbit RDye^® 680RD (926–68071) and goat anti-mouse RDye^® 800CW (926–32210), were from LICOR. Anti-pY45-STAT3 and anti-acetyl-K78-STAT3 polyclonal antibodies were prepared by AB-land, Inc. (Hangzhou, China).
Recombinant human IL-6 was from Life Technologies, LPS was from Sigma and recombinant human LIF was from Millipore. The dual-luciferase reporter assay system kit (E1910) was obtained from Promega.

Immunoprecipitation and cell lysis was performed in the following buffer – 50 mM HEPES pH7.5, 150 mM NaCl, 0.1% CHAPS, 50 mM L-arginine, 50 mM L-glutamic acid, 0.05% Tween, 50 mM NaF, 2 mM Na₃VO₄, 60 mM β-glycerophosphate di-sodium salt, 5 mM N-ethylmaleimide, 1 mM PMSF, 10 μM Z-LLF 1 mM DTT and 1× protease inhibitor without EDTA (Complete, Mini; Roche), with the addition of 150 mM NaCl (300 mM final concentration) and 100 mM KCl for washes. 6×10⁸ cells, harvested at a density of 2×10⁶/ml, were used per immunoprecipitation. Protein A dynal beads (Life Technologies) were used and the cell lysis solution was pre-cleared for 10 min with a bead-only slurry. Cleared extracts were incubated with GFP monoclonal antibody (clones 7.1 and 13.1, Roche) pre-coated dynal A beads for 30 min at 4°C and washed five times prior to elution from beads in loading buffer at 70°C for 15 min.